Using retroviral transduced bone marrow, several labs have shown that NUP98-HOXA9, NUP98-HHEX, NUP98-NSD1, and NUP98-TOP1 fusions are leukemogenic, and NUP98-HOXA9 and NHD13 fusions have been shown to be been shown to be leukemogenic in genetically engineered mice. However, a NUP98-TOP1 fusion was at best weakly oncogenic when expressed from a Vav promoter in transgenic mice. In order to better understand the leukemogenicity of NUP98 fused to non-HOX genes, we will study mice that express either a NUP98-RAP1GDS (NRG) or NUP98-PHF23 (NP23) fusion in hematopoietic cells using Vav regulatory elements to direct expression in the hematopoietic compartment. NRG mice were generated and transmitted the transgene in expected Mendellian ratio, and were documented to express the transgene. However, we could discern no hematopoietic abnormalities in these mice. NP23 mice have been generated, and we have followed a large cohort of offspring from two founders. Almost 100% of these mice develop leukemia within 1 year of life. Interestingly, the leukemic phenotype is very broad, including T and B cell leukemias, myeloid leukemias, and erythroid leukemias. We have begun a series of studies, including chromatin immunoprecipitation and gene expression profiling to identify targets of the NP23 fusion. Using Vav regulatory elements, we demonstrated that expression of a CALM-AF10 fusion predisposed mice to AML. However, the majority of patients with CALM-AF10 fusions develop not AML, but a gamma/delta pre-T lymphoblastic leukemia/lymphoma (pre-T LBL). Although we were able to show expression of CALM-AF10 mRNA in gamma/delta T-cells, it is possible that the expression level was inadequate to induce pre-T LBL, or that expression of CALM-AF10 led to AML before a pre-T LBL developed. Therefore, we plan to generate mice that express CALM-AF10 exclusively in gamma/delta T-cells. Insertion of an IRES and GFP reporter into the mouse Tcrd locus led to production of bi-cistronic transcripts encoding both Tcrd and GFP proteins, enabling the authors to track gamma/delta T-cells in vivo81. We obtained the vector used for those studies, and will replace the GFP cDNA with a CALM-AF10 cDNA. This vector will be used to target ES cells, and knock-in ES cells will be used to generate mice that express CALM-AF10 under the control of endogenous Tcrd regulatory elements. Unfortunately, we have encountered technical difficulties in generating this vector, and have been unable to generate any mice to complete this goal. Recent studies have demonstrated that HOXA9 is an important gene for stem cell self-renewal and is one of the most differentially expressed genes in patients with AML and MDS. Indeed, Hoxa cluster genes, especially Hoxa7/9/10, were among the most differentially expressed genes in the NHD13 and CALM-AF10 mice described in previous projects, suggesting that Hoxa9 is an important target for leukemic transformation. Therefore, we have generated mice that express Hoxa9 in hematopoietic cells, using Vav regulatory elements to enable us to compare these mice to the NHD13 mice. Some of the potential founders did not transmit the transgene, despite having greater than 30 pups genotyped, suggesting that the transgene may have been embryonic lethal in these mice. We have euthanized mice with timed pregnancies, and were able to document embryos that were transgenic, further supporting the possibility that the transgene was embryonic lethal in some founders. Two founders were able to transmit the transgene;however, they only expressed levels of the Hoxa9 transgene that were only slightly higher than wild-type controls. We have recently tested the ability of SCL, LMO1, and/or SV40 Large T antigen (TAg) to cause leukemia in zebrafish. The rationale for studying zebrafish is that they have a short generation time, high fecundity, small size, large, visible eggs, and visible, ex vivo development;development of T-lymphoid leukemia in fish would also allow comparative genomic approaches to determine common abnormalities and pathways among humans, mice, and fish. We used an Lck promoter from a related teleost fish (Fugu rubripes) to drive expression of genes in the developing fish thymus, and established transgenic lines for SCL, LMO1, and TAg. After 3 years of study, there is no evidence that any of these lines develop lymphoid malignancies. However, the SCL, LMO1, and TAg lines have decreased survival and a markedly increased incidence of seminoma (testicular germ cell tumor). Preliminary studies indicate the following;analysis of larger cohorts is currently in progress. Two different Lck-TAg lines have been followed, and have a cumulative incidence of seminoma of 12 or 16% by 36 months of life. Transgenic SCL and LMO1 fish are also predisposed to seminoma, with a cumulative incidence of 25 and 14 % by 36 months of life. The seminomas showed variable contribution of spermatocyte, spermatid, and spermatogonial components, and expression of genes that have been used as markers for human (AP2alpha, OCT4) or fish (Sox9a, Vas, Wt1) testicular tumors. We were puzzled by these findings, as we anticipated that the Lck promoter would direct expression exclusively in the thymus. However, the Fugu Lck promoter was promiscuous, and we detected TAg expression in seminomas and testes. A manuscript describing these fidings has recently been submitted.